Bottom Line:
Here, to better determine these affinities we have measured Ca(2+) dose-response curves of channel activity at constant voltage for the wild-type mSlo channel (minus its low-affinity Ca(2+) binding site) and for channels that have had one or the other Ca(2+) binding site disabled via mutation.To accurately determine these dose-response curves we have used a series of 22 Ca(2+) concentrations, and we have used unitary current recordings, coupled with changes in channel expression level, to measure open probability over five orders of magnitude.Our results indicate that at -80 mV the Ca(2+) bowl has higher affinity for Ca(2+) than does the RCK1 site in both the opened and closed conformations of the channel, and that the binding of Ca(2+) to the RCK1 site is voltage dependent, whereas at the Ca(2+) bowl it is not.

ABSTRACTIt has been established that the large conductance Ca(2+)-activated K(+) channel contains two types of high-affinity Ca(2+) binding sites, termed the Ca(2+) bowl and the RCK1 site. The affinities of these sites, and how they change as the channel opens, is still a subject of some debate. Previous estimates of these affinities have relied on fitting a series of conductance-voltage relations determined over a series of Ca(2+) concentrations with models of channel gating that include both voltage sensing and Ca(2+) binding. This approach requires that some model of voltage sensing be chosen, and differences in the choice of voltage-sensing model may underlie the different estimates that have been produced. Here, to better determine these affinities we have measured Ca(2+) dose-response curves of channel activity at constant voltage for the wild-type mSlo channel (minus its low-affinity Ca(2+) binding site) and for channels that have had one or the other Ca(2+) binding site disabled via mutation. To accurately determine these dose-response curves we have used a series of 22 Ca(2+) concentrations, and we have used unitary current recordings, coupled with changes in channel expression level, to measure open probability over five orders of magnitude. Our results indicate that at -80 mV the Ca(2+) bowl has higher affinity for Ca(2+) than does the RCK1 site in both the opened and closed conformations of the channel, and that the binding of Ca(2+) to the RCK1 site is voltage dependent, whereas at the Ca(2+) bowl it is not.

Mentions:
The BKCa channel is both Ca2+ and voltage sensitive, and the effects of these stimuli are often displayed as a series of G-V relations determined at several Ca2+ concentrations (Barrett et al., 1982). Such a series, determined from BKCa channels exogenously expressed in TSA-201 cells, is shown in Fig. 1 B. The data are from excised inside-out macropatches (Fig. 1 A). Increasing intracellular Ca2+ shifts the channel's G-V curve leftward, an effect that is known under wild-type conditions to be due to three types of Ca2+ binding sites, two of high affinity and one of low affinity. The channels in the patches of Fig. 1, however, contained the mutation E399N, which eliminates low-affinity Ca2+ sensing (Shi et al., 2002; Xia et al., 2002) and thereby allows one to examine high-affinity Ca2+ sensing in isolation. We refer to the mouse Slo (mSlo) channel carrying this mutation as ΔE. Increasing Ca2+ from 3 nM to 2.5 mM shifts the ΔE G-V relation ∼−200 mV.

Mentions:
The BKCa channel is both Ca2+ and voltage sensitive, and the effects of these stimuli are often displayed as a series of G-V relations determined at several Ca2+ concentrations (Barrett et al., 1982). Such a series, determined from BKCa channels exogenously expressed in TSA-201 cells, is shown in Fig. 1 B. The data are from excised inside-out macropatches (Fig. 1 A). Increasing intracellular Ca2+ shifts the channel's G-V curve leftward, an effect that is known under wild-type conditions to be due to three types of Ca2+ binding sites, two of high affinity and one of low affinity. The channels in the patches of Fig. 1, however, contained the mutation E399N, which eliminates low-affinity Ca2+ sensing (Shi et al., 2002; Xia et al., 2002) and thereby allows one to examine high-affinity Ca2+ sensing in isolation. We refer to the mouse Slo (mSlo) channel carrying this mutation as ΔE. Increasing Ca2+ from 3 nM to 2.5 mM shifts the ΔE G-V relation ∼−200 mV.

Bottom Line:
Here, to better determine these affinities we have measured Ca(2+) dose-response curves of channel activity at constant voltage for the wild-type mSlo channel (minus its low-affinity Ca(2+) binding site) and for channels that have had one or the other Ca(2+) binding site disabled via mutation.To accurately determine these dose-response curves we have used a series of 22 Ca(2+) concentrations, and we have used unitary current recordings, coupled with changes in channel expression level, to measure open probability over five orders of magnitude.Our results indicate that at -80 mV the Ca(2+) bowl has higher affinity for Ca(2+) than does the RCK1 site in both the opened and closed conformations of the channel, and that the binding of Ca(2+) to the RCK1 site is voltage dependent, whereas at the Ca(2+) bowl it is not.

ABSTRACTIt has been established that the large conductance Ca(2+)-activated K(+) channel contains two types of high-affinity Ca(2+) binding sites, termed the Ca(2+) bowl and the RCK1 site. The affinities of these sites, and how they change as the channel opens, is still a subject of some debate. Previous estimates of these affinities have relied on fitting a series of conductance-voltage relations determined over a series of Ca(2+) concentrations with models of channel gating that include both voltage sensing and Ca(2+) binding. This approach requires that some model of voltage sensing be chosen, and differences in the choice of voltage-sensing model may underlie the different estimates that have been produced. Here, to better determine these affinities we have measured Ca(2+) dose-response curves of channel activity at constant voltage for the wild-type mSlo channel (minus its low-affinity Ca(2+) binding site) and for channels that have had one or the other Ca(2+) binding site disabled via mutation. To accurately determine these dose-response curves we have used a series of 22 Ca(2+) concentrations, and we have used unitary current recordings, coupled with changes in channel expression level, to measure open probability over five orders of magnitude. Our results indicate that at -80 mV the Ca(2+) bowl has higher affinity for Ca(2+) than does the RCK1 site in both the opened and closed conformations of the channel, and that the binding of Ca(2+) to the RCK1 site is voltage dependent, whereas at the Ca(2+) bowl it is not.